Expressed Protein Selenoester Ligation

Abstract Herein, we describe the development and application of a novel expressed protein selenoester ligation (EPSL) methodology for the one‐pot semi‐synthesis of modified proteins. EPSL harnesses the rapid kinetics of ligation reactions between modified synthetic selenopeptides and protein aryl selenoesters (generated from expressed intein fusion precursors) followed by in situ chemoselective deselenization to afford target proteins at concentrations that preclude the use of traditional ligation methods. The utility of the EPSL technology is showcased through the efficient semi‐synthesis of ubiquitinated polypeptides, lipidated analogues of the membrane‐associated GTPase YPT6, and site‐specifically phosphorylated variants of the oligomeric chaperone protein Hsp27 at high dilution.


Table of Contents
General peptide synthesis procedures 4 General procedure for acyl hydrazide to selenoester conversion 6 Ubiquitin acyl hydrazide to selenoester conversion 6 General procedure for one-pot selenoesterification, ligation and deselenization (EPSL) 11 Synthesis of Ubiquitin-TMEM43 conjugate 11 Synthesis of Ubiquitin-ZAP70 conjugate 14 Semi-synthesis of lipidated analogues of YPT6 18 Folding of semi-synthetic lipidated analogues of YPT6 28 Semi-synthesis of Hsp27 phosphoforms 30 Folding and CD spectra of Hsp27 phosphoforms 42 Chaperone assay for Hsp27 phosphoforms 43

Materials
Peptide grade dimethylformamide (DMF) was obtained from Labscan. Amino acids, coupling reagents and resins for Fmoc-solid-phase peptide synthesis (SPPS) were obtained from either Novabiochem, GL Biochem or Sigma Aldrich. Manual SPPS was performed in polypropylene syringes equipped with Teflon filters, purchased from Torviq. Analytical reversed-phase ultraperformance liquid chromatography (RP-UPLC) was performed on either a Waters Acquity UPLC system equipped with PDA eλ detector (λ = 210 -400 nm), Sample Manager FAN and Quaternary Solvent Manager (H-class) modules or a Waters System 2695 separations module with a 2996 photodiode array detector. Peptides were analyzed using an XBridge BEH 5 µm, 2.1 x 150 mm wide-pore column (C18) at a flow rate of 0.7 mL min -1 or 1 mL min -1 on the HPLC system or Waters Acquity UPLC BEH 1.7 µm 2.1 x 50 mm column (C18) at a flow rate of 0.6 mL min -1 on the UPLC system. Gradients for the UPLC system were run over five minutes with an initial 1 min equilibration step (i.e. gradient from 1-6 min) while the gradients for the HPLC system were run for 30 min with an initial 5 min equilibration step (i.e. gradient from 5-35 min). Both instruments were run using a mobile phase composed of 0.1% trifluoroacetic acid (TFA) in H2O (Solvent A) and 0.1% trifluoroacetic acid in acetonitrile (Solvent B) in a linear gradient as indicated. Analysis of the chromatograms was conducted using Empower 3 Pro software (2010) and retention times (Rt min) of pure peptides and proteins are reported with the gradients specified.
Preparative and semi-preparative reversed-phase HPLC was performed using a Waters 600E Multisolvent Delivery System with a Rheodyne 7725i injection valve (5 mL loading loop) and Waters 500 pump with a Waters 490E programmable wavelength detector operating at 214, 230, 254 or 280 nm. Preparative reversed-phase HPLC was performed using a Waters Sunfire C18 column (5 μm, 19 × 250 mm) at a flow rate of 14 mL min −1 . Semi-preparative reversed-phase HPLC was performed using either a Waters XBridge-BEH300 wide-pore C18 column (5 μm, 10 × 250 mm) or Symmetry C4 column (300 Å, 5 μm, 10 mm × 250 mm) at a flow rate of 4 mL min −1 or (300 Å, 5 μm, 2.1 mm × 150 mm) at a flow rate of 0.7 mL min −1 . Ubiquitin conjugate 5 was purified on a Phenomenex Luna C18 column (100 Å, 5 μm, 10 × 250 mm) heated to 50 °C using a Waters column heater module at a flow rate of 4 mL min −1 , with the gradients as described. A mobile phase of 0.1% trifluoroacetic acid in water (Solvent A) and 0.1% trifluoroacetic acid in acetonitrile (Solvent B) was used in all other cases, using the linear gradients specified. After lyophilization, peptides were isolated as trifluoroacetate salts.
LC-MS was performed either on a Shimadzu LC-MS 2020 instrument consisting of a LC-M20A pump and a SPD-20A UV/Vis detector coupled to a Shimadzu 2020 mass spectrometer (ESI) operating in positive mode, or a Shimadzu UPLC-MS equipped with the same modules as the LC-MS system except for a SPD-M30A diode array detector. Separations were performed on the LC-MS system either on a Waters Sunfire 5 µm, 2.1 x 150 mm column (C18), or wide-pore equivalent operating at a flow rate of 0.7 mL min -1 or 1 mL min -1 . Separations on the UPLC-MS system were performed using a Waters Acquity UPLC BEH 300Å 1.7 µm 2.1 x 50 mm column (C18) at a flow rate of 0.6 mL min -1 . Separations were performed using a mobile phase of 0.1% formic acid in water (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B) and a linear gradient of 0-50% B over 30 min on the LC-MS System and 0-60% B over 8 min on the UPLC-MS system. agitated at room temperature. Upon complete PMB deprotection (as judged by UPLC-MS), the reaction mixture was diluted with water and purified by RP-HPLC to afford the corresponding peptide diselenide dimer.
General procedure for acyl hydrazide to selenoester conversion: The following describes the general optimized procedure for selenoester formation under which all conversions were conducted unless otherwise stated.
A buffer solution containing 200 mM TCEP, 200 mM HEPES, 50 mM DPDS and 6 M Gnd.HCl was freshly prepared in MilliQ water and the pH was adjusted to 1.5-2.0 using 5 M aqueous HCl, followed by sparging of the solution with argon for 15-20 min. The peptide or protein acyl hydrazide substrate was then dissolved in the buffer (to final concentrations of 250 µM), followed by the addition of 5 eq. of acetylacetone (acac) from a 150 mM aqueous stock. The solution was then allowed to stir at room temperature under an atmosphere of argon for 2-3 h. Completion of the selenoesterification reaction was monitored by UPLC-MS followed by Et2O extraction of residual DPDS. The crude reaction mixture was either subjected directly to a DSL reaction (in the case of the EPSL methodology) or subjected to HPLC purification and lyophilization to afford the purified peptide selenoester as a white fluffy solid.

Recombinant expression and purification of Ubiquitin-hydrazide:
The DNA sequence of human ubiquitin (Ub) was amplified by PCR (primer sequences attached) and subsequently cloned upstream of the Mycobacterium xenopi DNA Gyrase A (Mxe GyrA) intein, a His7 tag and a chitin-binding domain (CBD) into a pTXB1 (New England Biolabs) plasmid via NdeI and SpeI restriction sites. Protein expression was performed using the E. coli BL21(DE3) Gold (Agilent) strain and 2YT medium (16 g/L Trypton, 10 g/L Yeast extract, 5 g/L NaCl) containing 100 µg/mL ampicillin at 37 °C.
Overnight cultures were diluted to OD600 0.2, grown until OD600 0.7 and protein overexpression was induced with 1 mM IPTG. After 2 h, the culture was centrifuged for 20 min at 10,000 g, the cell pellets were resuspended in TBS buffer (50 mM Tris, 150 mM NaCl, pH 8) and lysed twice

General procedure for one-pot selenoesterification, ligation and deselenization (EPSL)
The protein acyl hydrazide substrate was converted to the corresponding selenoester according to the general procedure above. The peptide diselenide dimer fragment was then added to the crude reaction mixture and the pH carefully raised to pH 5 using 5 M NaOH(aq) and the reaction was allowed to proceed at room temperature, with completion of the reaction assessed by UPLC-MS. Following completion of the ligation (typically in 40-50 min), Et2O extraction (x10) was performed to remove residual DPDS. Solid TCEP (40 mM) and reduced glutathione (40 mM) were then added and the pH of resulting solution was adjusted to 7.0 followed by thorough sparging with argon. The reaction mixture was incubated at room temperature or 37 o C for 16 h as specified in the experimental procedure.
After centrifugation, the supernatant was concentrated in an Amicon Ultra-0.5 device with a 10 kDa molecular weight cut-off and analyzed via RP-HPLC by isocratic elution with 50 mM potassium phosphate buffer at pH 7 containing 10 mM tetrabutylammonium bromide (TBAB) and 8% MeCN. Absorption of nucleotides was followed at 256 nm.
For the folding of dodecylated YPT6 (6) at 50 µM initial concentration, no signal for nucleotide bound to the protein after dialysis and concentration was detected. However, analysis of the precipitate collected after dialysis by SDS-PAGE revealed that dodecylated YPT6 (6) was precipitated ( Figure S16). Higher molecular weight species also indicate irreversible aggregation. Palmitylated YPT6 (7) was dissolved at 10 µM initial concentration and no precipitated protein was detected after folding and centrifugation. Analysis of the soluble protein revealed a mixture of GTP and GDP bound to the protein, indicating folding ( Figure S17). The ratio of GDP to GTP is increased due to the intrinsic, low GTPase activity of YPT6.

Semi-synthesis of Hsp27 phosphoforms: Recombinant Expression and Purification of Hsp27(1-172) hydrazide (15):
The Hsp27(1-172)-intein fusion protein was expressed from an already available plasmid pTXB3-Hsp27-Mxe-CBD containing the human Hsp27(1-172) DNA sequence followed by the Mxe GyrA intein, a His7 tag and a chitin binding domain. [6] Protein expression was performed in E. coli BL21(DE3) Rosetta 2 grown in 2YT medium (16 g/L Trypton, 10 g/L Yeast extract, 5 g/L NaCl) containing 100 µg/mL ampicillin and 30 µg/mL chloramphenicol at 37 °C. Overnight cultures were diluted to OD600 0.2, grown until OD600 0.7 and protein overexpression was induced by addition of 1 mM IPTG. After 4 h, the culture was centrifuged for 20 min at 10,000 g, the cell pellets were resuspended in TBS buffer (50 mM Tris, 150 mM NaCl, pH 7.5) and lysed twice in a high-pressure cell disrupter (Constant Systems). The lysate was centrifuged for 30 min at 50,000 g to separate soluble and insoluble fractions. The overexpressed Hsp27 intein fusion construct was isolated from the insoluble fraction (inclusion body) by dissolving the pellet obtained from 2 L culture in 25 mL of solubilization buffer (6 Gnd·HCl, 50 mM Tris, pH 7.5).

MTERRVPFSLLRGPSWDPFRDWYPHSRLFDQAFGLPRLPEEWSQWLG GSSWPGYVRPLPPAAIESPAVAAPAYSRALSRQLSSGVSEIRHTADRWR VSLDVNHFAPDELTVKTKDGVVEITGKHEERQDEHGYISRCFTRKYTLPP GVDPTQVSSSLSPEGTLTVEAPMPKL
Hsp27 ( 22+ . ESI-MS data was collected over the entire gradient and wash cycle of the UPLC-MS; [* = Masses correspond to partial removal of the N-terminal methionine (MWMet = 131 Da) in the expressed Hsp27 (1-172)-NHNH2. This is often observed for proteins overexpressed in bacteria and has no functional consequences]. [6] (C) Deconvoluted mass spectrum of 15; The masses correspond to the presence and absence of the Nterminal methionine derived from the expressed Hsp27 segment (vide supra).
CD spectra were recorded using a Chirascan Plus CD-spectrophotometer (Applied Photophysics, United Kingdom). CD spectra were recorded at 25 °C from 200 to 260 nm in 1 nm steps. For each spectrum, 10 measurements were averaged and the background (buffer only) was subtracted. The raw data were exported from Pro-Data software as CSV and further processed using OriginPro.
The assay was performed according to the literature method with minor modifications. [7] Citrate Synthase (CS) from porcine heart was purchased from Sigma-Aldrich (Taufkirchen, Germany) as an ammonium sulfate suspension then centrifuged to remove most of the ammonium sulfate salts and dialyzed against the storage buffer (50 mM Tris·HCl, 2 mM EDTA, pH 8), final concentration 20-30 µM. The accurate concentration was then determined using bicinchoninic acid (BCA) assay (MW of CS = 48,969 Da) and this stock solution was flash frozen into liquid nitrogen in small aliquots (200-500 µL) and stored at -80 °C. Amorphous aggregation of CS was monitored via measuring the absorbance at 400 nm in a SAFAS UVmc2 double-beam UV-Vis spectrophotometer equipped with a temperature controlled multi-cell holder (SAFAS, Monaco) in 700 µL quartz cuvettes (Hellma Analytics, Germany), 600 µL final volume in triplicate. CS stock solution (obtained as above) was diluted with 40 mM HEPES·KOH (pH 7.5) to a final concentration of 2 µM, and the resulting solution was used as such (control) or treated with Hsp27 variants (0.45 µM final concentration) followed by incubation at 45 °C while measuring the absorbance at 400 nm over 45 minutes (600 µL final volume in triplicate). Prior to the addition, all Hsp27 variants (unmodified recombinant material and synthetic phosphorylated variants as lyophilized powders) were dissolved into 6 M guanidine hydrochloride in 40 mM HEPES·KOH (pH 7.5) buffer to a concentration of approx. 1.5 mg/mL, followed by dialysis against 40 mM HEPES·KOH (pH 7.5) buffer, overnight. The accurate concentration of these primary stocks and that of CS were determined via BCA assay and another stock of all Hsp27 samples of concentration 1 mg/mL was prepared. A baseline correction employing only the assay buffer (40 mM HEPES·KOH, pH 7.5) was also performed. The raw data were exported from SAFAS software as a Microsoft Excel worksheet and processed using Microsoft Excel and OriginPro. The results were expressed as average relative UV absorbance at 400 nm, where relative absorption at 400 nm = (absorption at 400 nm)/(maximal absorption at 400 nm by aggregating CS in the absence of a chaperone).

H and 13 C NMR Spectra (building blocks Sand S2):
Building block S1